title: Bimodal voltage dependence of TRPA1: mutations of a key pore helix residue reveal strong intrinsic voltage-dependent inactivation.
abstract: Transient receptor potential A1 (TRPA1) is implicated in somatosensory processing and pathological pain sensation. Although not strictly voltage-gated, ionic currents of TRPA1 typically rectify outwardly, indicating channel activation at depolarized membrane potentials. However, some reports also showed TRPA1 inactivation at high positive potentials, implicating voltage-dependent inactivation. Here we report a conserved leucine residue, L906, in the putative pore helix, which strongly impacts the voltage dependency of TRPA1. Mutation of the leucine to cysteine (L906C) converted the channel from outward to inward rectification independent of divalent cations and irrespective to stimulation by allyl isothiocyanate. The mutant, but not the wild-type channel, displayed exclusively voltage-dependent inactivation at positive potentials. The L906C mutation also exhibited reduced sensitivity to inhibition by TRPA1 blockers, HC030031 and ruthenium red. Further mutagenesis of the leucine to all natural amino acids individually revealed that most substitutions at L906 (15/19) resulted in inward rectification, with exceptions of three amino acids that dramatically reduced channel activity and one, methionine, which mimicked the wild-type channel. Our data are plausibly explained by a bimodal gating model involving both voltage-dependent activation and inactivation of TRPA1. We propose that the key pore helix residue, L906, plays an essential role in responding to the voltage-dependent gating.
authors.affiliation: Department of Clinical Pharmacology, the First Affiliated Hospital, Chongqing Medical University, Chongqing, China.
authors.forename: Xia
authors.initials: X
authors.lastname: Wan
authors.forename: Yungang
authors.initials: Y
authors.lastname: Lu
authors.forename: Xueqin
authors.initials: X
authors.lastname: Chen
authors.forename: Jian
authors.initials: J
authors.lastname: Xiong
authors.forename: Yuanda
authors.initials: Y
authors.lastname: Zhou
authors.forename: Ping
authors.initials: P
authors.lastname: Li
authors.forename: Bingqing
authors.initials: B
authors.lastname: Xia
authors.forename: Min
authors.initials: M
authors.lastname: Li
authors.forename: Michael X
authors.initials: MX
authors.lastname: Zhu
authors.forename: Zhaobing
authors.initials: Z
authors.lastname: Gao
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D007930:Leucine / Q000235:genetics; D008564:Membrane Potentials; D051379:Mice; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary; D000074025:TRPA1 Cation Channel; D050051:Transient Receptor Potential Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000074025:TRPA1 Cation Channel; D050051:Transient Receptor Potential Channels; C475685:Trpa1 protein, mouse; D007930:Leucine